1. Field of the Invention
This invention relates to the fields of chemistry and medicine. More particular, the invention relates to compounds and compositions and use of compounds and compositions as therapeutic agents, including antimicrobial agents and Efflux Pump Inhibitors (EPIs).
2. Description of the Related Art
Antibiotics have been effective tools in the treatment of infectious diseases during the last half-century. From the development of antibiotic therapy to the late 1980s, there was almost complete control over bacterial infections in developed countries. However, in response to the pressure of antibiotic usage, multiple resistance mechanisms have become widespread and are threatening the clinical utility of antibacterial therapy. The increase in antibiotic resistant strains has been particularly common in major hospitals and care centers. The consequences of the increase in resistant strains include higher morbidity and mortality, longer patient hospitalization, and an increase in treatment costs.
The emergence of resistant bacterial strains is considered to be the number one infectious disease threat in hospitals and costs the U.S. Healthcare system $5 billion per year. Infections that were once treatable with antibiotics are becoming difficult and in some cases impossible to treat. As a result, 2 million people in the U.S. are infected by a bacterial pathogen while in the hospital each year, of which approximately 90,000 die, and the majority incur prolonged hospital and/or outpatient antibiotic therapy at significant pharmacoeconomic cost. More than 70% of the bacteria responsible for these infections are resistant to at least one of the antibiotics commonly used to fight them. Therefore the prevention of resistance is considered to be a high priority among infectious disease clinicians.
Resistance is not an isolated problem for specific antibiotics, but a global one affecting all antibiotics. Numerous studies have documented a strong correlation between the increased use of antibiotics and an increase in bacterial resistance, which can sometimes occur even during therapy. For example, one study documented a growth in fluoroquinolone resistance in Pseudomonas aeruginosa from 25% in 1997 to 33% in 2002. Another study reported that Pseudomonas aeruginosa resistance to fluoroquinolones increased from 29% in 1999 to 38% in 2001, with some hospitals reporting resistance rates as high as 60%. The level of fluoroquinolone use is directly correlated to the level of resistance, thus explaining the variability of resistance in different hospitals. Infections caused by resistant strains and or by strains that develop resistance during therapy limit the use of what are otherwise safe and effective fluoroquinolones as first-line therapy in hospitals.
Bacteria have developed several different mechanisms to overcome the action of antibiotics. These mechanisms of resistance can be specific for a molecule or a family of antibiotics, or can be non-specific and be involved in resistance to unrelated antibiotics. Several mechanisms of resistance can exist in a single bacterial strain, and those mechanisms may act independently or they may act synergistically to overcome the action of an antibiotic or a combination of antibiotics. Specific mechanisms include degradation of the drug, inactivation of the drug by enzymatic modification, and alteration of the drug target. There are, however, more general mechanisms of drug resistance, in which access of the antibiotic to the target is prevented or reduced by decreasing the transport of the antibiotic into the cell or by increasing the efflux of the drug from the cell to the outside medium. Both mechanisms can lower the concentration of drug at the target site and allow bacterial survival in the presence of one or more antibiotics that would otherwise inhibit or kill the bacterial cells. Some bacteria utilize both mechanisms, combining a low permeability of the cell wall (including membranes) with an active efflux of antibiotics.
In recent years interest in efflux-mediated resistance in bacteria has been triggered by the growing amount of data implicating efflux pumps in clinical isolates. The phenomenon of antibiotic efflux was first discovered in 1980, in the context of the mechanism of tetracycline resistance in enterobacteria. Since then, it has been shown that efflux of antibiotics can be mediated by more than one pump in a single organism and that almost all antibiotics are subject to resistance by this mechanism.
Some efflux pumps selectively extrude specific antibiotics. Examples of such pumps include the Tet or CmlA transporters, which can extrude tetracycline or chloramphenicol, respectively. Other efflux pumps, so-called multi-drug resistance (MDR) pumps, extrude a variety of structurally diverse compounds. In the latter case, a single efflux system may confer resistance to multiple antibiotics with different modes of action. In this respect, bacterial MDR pumps are similar to mammalian MDR transporters. In fact, one such pump, P-glycoprotein, the first discovered MDR pump, confers multiple drug resistance on cancer cells and is considered to be one of the major reasons for tumor resistance to anti-cancer therapy. A typical example of bacterial MDR pump is MexAB-OprM from Pseudomonas aeruginosa. This pump has been shown to affect the susceptibility of the organism to almost all antibiotic classes which fluoroquinolones, β-lactams, macrolides, phenicols, tetracyclines, and oxazolidinones.
Efflux pumps in gram-positive bacteria excrete their substrates across a single cytoplasmic membrane. This is also the case for some pumps in gram-negative bacteria, and as a result their substrates are effluxed into the periplasmic space. Other efflux pumps from gram-negative bacteria efflux their substrates directly into the external medium, bypassing the periplasm and the outer membrane. These pumps are organized in complex three component structures, which traverse both inner and outer membranes. They consist of a transporter located in the cytoplasmic membrane, an outer membrane channel and a periplasmic ‘linker’ protein, which brings the other two components into contact. It is clearly advantageous for gram-negative bacteria to efflux drugs by bypassing the periplasm and outer membrane. In gram-negative bacteria the outer membrane significantly slows down the entry of both lipophilic and hydrophilic agents. The former, such as erythromycin and fusidic acid, are hindered by the lipopolysaccharide components of the outer leaflet of the outer membrane bilayer. Hydrophilic agents cross the outer membrane through water-filled porins whose size prevents rapid diffusion, even for small compounds such as fluoroquinolones and some β-lactams. Thus, direct efflux creates the possibility for two different mechanisms to work synergistically to provide the cell with a potent defense mechanism. Furthermore, direct efflux into the medium leads to decreased amounts of drugs not only in the cytoplasmic but also in the periplasmic space. This could explain the apparently paradoxical finding that efflux pumps protect gram-negative bacteria from β-lactam antibiotics whose target penicillin-binding proteins are found in the periplasm.
Many MDR pumps are encoded by the genes, which are normal constituents of bacterial chromosomes. In this case increased antibiotic resistance is a consequence of over-expression of these genes. Thus bacteria have the potential to develop multi-drug resistance without the acquisition of multiple specific resistance determinants. In some cases, the simultaneous operation of efflux pumps and other resistance mechanisms in the same cell results in synergistic effects.
While some genes encoding efflux pumps are not expressed in wild type cells and require induction or regulatory mutations for expression to occur, other efflux genes are expressed constitutively. As a result, wild type cells have basal level of efflux activity. This basal activity of multi-drug efflux pumps in wild type cells contribute to intrinsic antibiotic resistance, or more properly, decreased antibiotic susceptibility. This intrinsic resistance may be low enough for the bacteria to still be clinically susceptible to therapy. However, the bacteria might be even more susceptible if efflux pumps were rendered non-functional, allowing lower doses of antibiotics to be effective. To illustrate, P. aeruginosa laboratory-derived mutant strain PAM1626, which does not produce any measurable amounts of efflux pump is 8 to 10 fold more susceptible to levofloxacin and meropenem than the parent strain P. aeruginosa PAM1020, which produces the basal level of MexAB-OprM efflux pump. Were it not for efflux pumps, the spectrum of activity of many so-called ‘gram-positive’ antibiotics could be expanded to previously non-susceptible gram-negative species. This can be applied to ‘narrow-spectrum’ β-lactams, macrolides, lincosamides, streptogramins, rifamycins, fusidic acid, and oxazolidinones—all of which have a potent antibacterial effect against engineered mutants lacking efflux pumps.
It is clear that in many cases, a dramatic effect on the susceptibility of problematic pathogens would be greatly enhanced if efflux-mediated resistance were to be nullified. Two approaches to combat the adverse effects of efflux on the efficacy of antimicrobial agents can be envisioned: identification of derivatives of known antibiotics that are not effluxed and development of therapeutic agents that inhibit transport activity of efflux pumps and could be used in combination with existing antibiotics to increase their potency.
There are several examples when the first approach has been successfully reduced to practice. These examples include new fluoroquinolones, which are not affected by multidrug resistance pumps in Staphylococcus areus or Streptococcus pneumonia or new tetracycline and macrolide derivatives, which are not recognized by the corresponding antibiotic-specific pumps. However, this approach appears to be much less successful in the case of multidrug resistance pumps from gram-negative bacteria. In gram-negative bacteria, particular restrictions are imposed on the structure of successful drugs: they must be amphiphilic in order to cross both membranes. It is this very property that makes antibiotics good substrates of multi-drug resistance efflux pumps from gram-negative bacteria. In the case of these bacteria the efflux pump inhibitory approach becomes the major strategy in improving the clinical effectiveness of existing antibacterial therapy.
Tigecycline is an antibiotic recently approved by the Food and Drug Administration. This antibiotic, which belongs to the glycylcycline class of tetracycline analogs, is not susceptible to the known resistance mechanisms affecting other tetracyclines. It is active against organisms with tetracycline resistant, modified ribosomes, and it avoids tetracycline-specific efflux pumps from gram-positive and gram-negative bacteria. However, multi-component multi-drug resistance (MDR) efflux pumps from gram-negative bacteria driven by RND-transporters, and some MDR pumps from gram-positive bacteria (such as MepA from the MATE-family) can efflux tigecycline with a resulting decrease in susceptibility. Due to the activity of such pumps, several species of bacteria are considered to be generally non-susceptible to tigecycline at all. Among such species are Pseudomonas, Proteus, Morganella, and Providentia. In addition, reports from the literature implicate RND-containing efflux pumps in low-level acquired tigecycline resistance in several bacterial species which are susceptible to this antibiotic. The non-limiting examples include Klebsiella, Enterobacter, E. coli, Citrobacter, and Staphylococcus. The frequency of emergence of efflux-mediated tigecycline resistance in Klebsiella pneumonia was reported to be ˜4×10−8.